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Human Molecular Genetics, 2003, Vol. 12, No. 15 1839-1845
DOI: 10.1093/hmg/ddg192
© 2003 Oxford University Press

Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblasts

Jan-Willem Taanman1,*, John R. Muddle1 and Ania C. Muntau2

1University Department of Clinical Neurosciences, Royal Free and University College Medical School, University College London, London, UK and 2Dr von Hauner Children's Hospital, Ludwig-Maximilians University, Munich, Germany

Received April 4, 2003; Revised May 15, 2003; Accepted May 24, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Deoxyguanosine kinase is a constitutively expressed, mitochondrial enzyme of the deoxyribonucleoside salvage pathway. Deficiency of deoxyguanosine kinase causes early-onset, hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome. To clarify the molecular mechanism of the disease, a skin fibroblast culture was studied from a patient carrying a homozygous nonsense mutation in the gene for deoxyguanosine kinase. In situ examination of DNA synthesis demonstrated that, although mtDNA synthesis is cell cycle independent in control fibroblasts, mtDNA synthesis occurs mainly during the S-phase in deoxyguanosine kinase-deficient cells. Consistent with this observation, it was found that the mtDNA content of exponentially growing, deoxyguanosine kinase-deficient cells is only mildly affected. When cycling is inhibited by serum-deprivation and cells are in a resting state, however, the mtDNA content drops considerably in deoxyguanosine kinase-deficient cells, yet remains stable in control fibroblasts. The decline in mtDNA content in resting, deoxyguanosine kinase-deficient cells can be prevented by dGMP and dAMP supplementation, providing conclusive evidence that substrate limitation triggers mtDNA depletion in deoxyguanosine kinase-deficient cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In mammals, the bulk of deoxyribonucleotides are synthesized de novo from low molecular weight precursors. Activities of enzymes of the de novo pathway are highest in the S-phase of the cell cycle and undetectable in quiescent or terminally differentiated cells resting in G0. Consequently, deoxynucleotide pool size variation closely correlates to the progression of cells in the cycle (1). Deoxyribonucleotides can also be provided by salvage of deoxyribonucleosides, arising from extra- or intracellular degradation of DNA. The first step of the salvage pathway is performed by the deoxyribonucleoside kinases which catalyse the phosphorylation of deoxyribonucleosides to the corresponding deoxyribonucleoside monophosphates. There are four mammalian deoxyribonucleoside kinases with overlapping substrate specificity: thymidine kinase 1 and 2 (EC 2.7.1.21), deoxycytidine kinase (EC 2.7.1.74) and deoxyguanosine kinase (EC 2.7.1.113). Both thymidine kinase 1 and 2 phosphorylate thymidine and deoxyuridine, although thymidine kinase 2 also uses deoxycytidine as a substrate. Deoxyguanosine kinase phosphorylates deoxyguanosine and deoxyadenosine. Deoxycytidine kinase also phosphorylates deoxyguanosine and deoxyadenosine but, in addition, is able to phosphorylate deoxycytidine. Thymidine kinase 1 and deoxycytidine kinase are cytosolic enzymes, whereas thymidine kinase 2 and deoxyguanosine kinase are located in the mitochondrial matrix (2,3). Deficiency of the mitochondrial enzymes has been linked to mitochondrial DNA (mtDNA) depletion syndrome (OMIM 251880).

More than 100 patients have been described with mtDNA depletion syndrome (4–15). This condition, which is characterized by a tissue-specific loss of mtDNA in association with defective mitochondrial respiratory chain function, is clinically heterogeneous. Most of the reported cases present shortly after birth with progressive liver failure and neurological abnormalities associated with lactic acidosis, hypoglycaemia and, more rarely, renal de Toni–Fanconi syndrome. Others present in infancy with myopathy associated with motor regression or a slowly progressive encephalomyopathy. In the early-onset form, the degree of mtDNA depletion is severe, with up to 99% reduction in affected tissues as compared with controls, and most patients die within their first year of life. In the late-onset form, depletion of mtDNA is less severe and survival is longer. The early-onset, hepatocerebral variant of mtDNA depletion syndrome has been ascribed to a mutation in the gene for deoxyguanosine kinase (DGUOK; OMIM 601465) (11), while the late-onset, myopathic variant has been ascribed to mutations in the gene for thymidine kinase 2 (TK2; OMIM 188250) (12). Genetic screening of a further 67 families with mtDNA depletion syndrome led to the identification of mutations in DGUOK or TK2 in another nine families (14–18).

In the present study, a fibroblast culture was characterized from a patient with hepatocerebral mtDNA depletion and deoxyguanosine kinase deficiency to investigate the molecular mechanism of the disease. It is shown that mtDNA synthesis occurs predominantly during the S-phase in patient cells, but is independent of the cell cycle in control cells. Furthermore, it is shown that mtDNA levels are only mildly affected and stable in exponentially growing patient fibroblasts, but deplete in resting patient fibroblasts. Depletion of mtDNA in these resting cells can be prevented by deoxyguanosine monophosphate (dGMP) and deoxyadenosine monophosphate (dAMP) supplementation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experiments with exponentially growing fibroblast cultures
A primary fibroblast culture was set up from a skin biopsy of a patient with a homozygous nonsense mutation in exon 3 of DGUOK (16). The proliferation rate of the patient culture was not markedly different from control fibroblast cultures. Levels of mtDNA in subsequent cell passages of the exponentially growing patient culture were compared with the levels of a control culture by Southern blot hybridization. DNA extracted from the cultures was digested with the restriction enzyme PvuII, which has a unique site in the circular mtDNA molecule. Blotted DNA was hybridized simultaneously with a probe for mtDNA and a probe for the multicopy, nuclear 18S rRNA gene to assess the amounts of DNA loaded (Fig. 1). Quantification of the hybridization signals by phosphorimaging revealed that mtDNA levels were stable in the control and the patient culture, albeit the mean mtDNA level (±SD) in the patient culture was 62%±8.2% (n=7) of the mean control level (100±16.9%, n=7).



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  		Figure 1. Levels of mtDNA present in increasing cell passages of a control and a patient fibroblast culture. Autoradiograms of Southern blots loaded with ~3 µg PvuII-digested, total genomic DNA per lane, and hybridized simultaneously with 32P-labelled probes for mtDNA and the nuclear 18S rRNA gene. The positions of the mtDNA and nuclear DNA (nDNA) signals are indicated.

 
Spectrophotometric enzyme activity measurements showed that cytochrome-c oxidase activities were stable in homogenates of subsequent cell passages of both the patient and control cultures. Relative to the amount of protein, the mean cytochrome-c oxidase activity of the patient culture was 77±36% (n=5) of the mean control activity (100±24%, n=5). Relative to the mitochondrial marker enzyme citrate synthase, the mean cytochrome-c oxidase activity of patient culture was 82±29% (n=5) of the mean control activity (100±38%, n=5).

To assess the expression of the mtDNA-encoded subunit I of cytochrome-c oxidase in individual cells, exponentially growing fibroblasts of the patient and a control were examined by immunocytochemistry. Cultures were first treated with Mitotracker dye to label the mitochondria fluorescent red, followed by fluorescent green immunostaining of cytochrome-c oxidase subunit I and fluorescent blue counterstaining of the nuclei with the DNA fluorchrome 4,6-diamidino-2-phenylindole (DAPI). Both cultures showed a uniform expression of subunit I in the mitochondria of all cells (Fig. 2A and B). A total of 144 microscopic fields were captured for each culture, and the mean grey levels were determined for both the green and red images of 2325 control and 875 patient cells within these fields. The distribution of the mean Mitotracker signal per cell was indistinguishable between the two cultures. The distribution of the mean subunit I signal per cell, however, showed a clear shift towards lower values for the patient cells compared with the control cells. The ratio of the two signals was calculated for each cell and is graphically displayed in Figure 3. Although not discernible by eye (Fig. 2A and B), the measurements revealed that most patient cells had a lower relative subunit I signal than the control cells (Fig. 3). The mean subunit I/Mitotracker ratio of the patient cells was 72±10% of that of the control cells (100±18%). Paired sample t-testing demonstrated that this difference was statistically significant (t-test mean difference: 19%; P<0.001). Subunit I expression levels of the patient cells did not change in subsequent cell passages compared with control cells (data not shown).



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  		Figure 2. Expression of cytochrome-c oxidase subunit I and synthesis of DNA in exponentially growing control and patient fibroblasts. (A, B) Pseudo-coloured, fluorescent micrographs of control (A) and patient (B) cells cultured in the presence of Mitotracker dye to label mitochondria red, followed by green immunostaining with anti-subunit I antibodies and blue nuclear counterstaining with DAPI. Note that subunit I is uniformly present in the mitochondria of all cells (red+green=yellow). (C, D) Pseudo-coloured, fluorescent micrographs of control (C) and patient (D) cells cultured in the presence of Br-dU, followed by green immunostaining with antibodies against incorporated Br-dU to visualize DNA synthesis. Nuclei were counterstained blue with DAPI. Note that all control cells show a punctate Br-dU incorporation pattern throughout their cytoplasm. In patient cells, this cytoplasmic staining is largely restricted to cells that show high levels of nuclear Br-dU incorporation (indicated with arrows). Bars: 20 µm.

 


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  		Figure 3. Relative cellular expression levels of cytochrome-c oxidase subunit I in exponentially growing control and patient fibroblast cultures. Patient and control cells were cultured in the presence of Mitotracker dye to label the mitochondria red, followed by green immunostaining with anti-subunit I antibodies and nuclear counterstaining with DAPI. The mean grey levels were measured for both the green and red images of 2325 control and 875 patient cells, and the ratio of the two mean signals was calculated for each cell. The histogram shows the frequency of cells with a particular subunit I/Mitotracker ratio at intervals of 0.05 for the patient (hatched bars) and control (solid bars) cultures. The total number of cells per culture is 100%.

 
Synthesis of nuclear and mtDNA was studied in patient and control fibroblasts by culturing the cells in the presence of the thymidine analogue 5-bromo-2'-deoxyuridine (Br-dU) for 6 h, followed by fluorescent green immunocytochemical detection of incorporated Br-dU and fluorescent blue counterstaining of the nuclei with DAPI. Image analysis of 381 patient and 463 control cells revealed that 28% of the patient and 46% of the control cells showed high levels of Br-dU incorporation in their nuclei, indicating that these were passing through S-phase during the labelling. In addition, all control cells showed a punctate Br-dU incorporation pattern throughout their cytoplasm (Fig. 2C). This cytoplasmic staining represents mtDNA synthesized during the Br-dU pulse (19,20). Patient fibroblasts in S-phase showed a cytoplasmic Br-dU incorporation pattern that was similar to the pattern in control cells, but most patients cells that were not in S-phase showed a markedly fainter Br-dU incorporation pattern than control cells (Fig. 2D). These Br-dU incorporation patterns did not change for the cultures with increasing cell passage number (data not shown). Reliable quantification of the cytoplasmic Br-dU signal was not possible, due to the halo of the brightly stained nuclei of cells in S-phase.

Experiments with quiescent fibroblast cultures
The Br-dU labelling experiments suggest that, in control cells, mtDNA synthesis is independent of the cell cycle, whereas, in patient cells, mtDNA synthesis occurs predominantly during S-phase. To investigate this further, we inhibited cell cycling of the cultures by serum withdraw. In addition, to determine the effects of exogenously added deoxypurine monophosphates on the mtDNA level of the patient culture, a batch of the serum-deprived patient cells was supplemented with 200 µM dGMP and 200 µM dAMP. Cultures were harvested 0–40 days after serum withdraw and Southern blot hybridization was used to determine mtDNA levels (Fig. 4A). Quantification of the hybridization signals revealed that mtDNA levels were stable in the control fibroblasts (Fig. 4B). At the beginning of the experiment, the mtDNA level in the patient cells was ~80% of that of the control, but decreased to ~20% of that of the control 28–40 days after serum withdraw (Fig. 4B). Levels of mtDNA, however, remained stable in patient cells that were supplemented with dGMP and dAMP (Fig. 4B). The experiment was repeated once, using a different control cell culture. Both experiments gave consistent results, except that the second control cell culture had slightly higher relative mtDNA levels than the first control culture.



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  		Figure 4. Levels of mtDNA present in control, patient and purine deoxyribonucleoside monophosphate (dRMP)-supplemented patient fibroblast cultures, 0–40 days after serum withdraw. (A) Autoradiogram of Southern blot loaded with ~3 µg PvuII-digested, total genomic DNA per lane, and hybridized simultaneously with 32P-labelled probes for mtDNA and the nuclear 18S rRNA gene. The positions of the mtDNA and nuclear DNA (nDNA) signals are indicated. (B) The phosphorimager-quantified, relative mtDNA content in the cultures (diamonds, control cells; squares, patient cells; triangles, patient cells supplemented with dGMP and dAMP). The mtDNA content in the control culture at t=0 was taken as 100%. Time after serum withdraw is indicated in days (d).

 
The expression levels of the mtDNA-encoded subunit II of cytochrome-c oxidase and the nuclear DNA-encoded flavoprotein of succinate dehydrogenase were assessed by western blotting in the serum-deprived cultures (Fig. 5A). Densitometric measurements of the protein signals revealed that the expression of the flavoprotein was similar for all three cultures and stable over time. Conversely, the subunit II signal in the patient cells was ~75% of that of the control at the beginning of the experiment. The subunit II signal decreased for all three cultures over time, but this decrease was more pronounced for the non-supplemented patient culture than for the control and the dGMP- and dAMP-supplemented patient cultures (Fig. 5B). The experiment was repeated twice, using different controls. The three experiments gave consistent results, except that the intensities of the protein signals of the second and third control cultures were slightly different from those of the first control culture.



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  		Figure 5. Levels of the flavoprotein of succinate dehydrogenase (SDH Fp) and subunit II of cytochrome-c oxidase (COX II) present in control, patient and purine deoxyribonucleoside monophosphate (dRMP)-supplemented patient fibroblast cultures, 0–40 days after serum withdraw. (A) Exposed film of western blot loaded with 8 µg protein extract per lane, and probed with a cocktail of monoclonal antibodies against SDH Fp and COX II. The migration of both proteins and molecular weight standards is indicated. (B) The densitometer-quantified, relative subunit II signals in the cultures (diamonds, control cells; squares, patient cells; triangles, patient cells supplemented with dGMP and dAMP). The subunit II signal in the control culture at t=0 was taken as 100%. Time after serum withdraw is indicated in days (d).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We studied a primary skin fibroblast culture from a patient harbouring a homozygous nonsense mutation in DGUOK. Mutations in this gene cause hepatocerebral mtDNA depletion syndrome (11,14,16). The mutation carried by our patient shortens the DGUOK reading frame by more than half. The patient's brother, who had the same homozygous mutation (16), exhibited severely depleted levels of mtDNA in liver (13). Although our patient is not expected to be able to synthesize a functionally active deoxyguanosine kinase, the mtDNA content of his exponentially growing fibroblasts was only mildly affected (Fig. 1). Image analysis of exponentially growing fibroblasts immunocytochemically stained for the mtDNA-encoded subunit I of cytochrome-c oxidase revealed that the subunit was uniformly expressed in the mitochondrial network of all patient cells (Fig. 2B). Most patient cells had, however, a lower subunit I staining intensity than control cells (Fig. 3). Enzymatic assays revealed a minor, non-significant effect on the cytochrome-c oxidase activity of the exponentially growing patient culture. Taken together, these findings suggest that the mitochondrial changes in the patient's exponentially growing fibroblast are relatively benign. This result is very different from our previous observations with primary cell cultures from patients with hepatocerebral mtDNA depletion syndrome in which we could not identify DGUOK mutations (16). Exponentially cultured cells from these patients showed a progressive loss of mtDNA, an intercellular mosaic expression of cytochrome-c oxidase subunit I and a marked cytochrome-c oxidase deficiency (6,7,9). These phenotypic differences suggest that the molecular mechanism which leads to hepatocerebral mtDNA depletion in these other patients is fundamentally different from the mechanism leading to hepatocerebral mtDNA depletion in deoxyguanosine kinase-deficient patients.

Br-dU is efficiently phosphorylated by thymidine kinase 1 and 2 (2,3) and incorporated into replicating DNA in place of thymidine. Br-dU is routinely used in immunocytochemical studies to identify cells in S-phase (21), and can also be used to visualize newly synthesized mtDNA (19,20). Immunocytochemistry of Br-dU-labelled, exponentially growing fibroblasts revealed a massive Br-dU incorporation in the nuclei of a fraction of the cells (Fig. 2C and D), indicating that these cells were progressing through S-phase during the Br-dU pulse. In addition, all control cells showed a punctate staining pattern in the cytoplasm, representing recently synthesized mtDNA (Fig. 2C). These results indicate that mtDNA synthesis occurs irrespective of the cell cycle in control fibroblasts. This is in agreement with earlier [3H]thymidine and Br-dU double-labelling experiments of mouse L-cell mtDNA, resolved on buoyant CsCl gradients, which suggested that mtDNA replication occurs at a constant rate throughout the cell cycle (22). In contrast, in the patient cells, mtDNA synthesis was cell cycle-dependent and occurred predominantly during S-phase (Fig. 2D). Deoxyribonucleotides synthesised in the cytosol are thought to be able to enter the mitochondria (23,24), a notion recently supported by the identification of two dedicated mitochondrial inner membrane carriers that efficiently import either deoxycytidine triphosphate (dCTP) (25), or deoxynucleoside diphosphates (26) into the mitochondrial matrix. As mtDNA synthesis is continuous, a steady supply of deoxynucleotides is crucial for maintenance of mtDNA copy numbers. Our Br-dU-labelling experiments suggest that, during S-phase, when the cytosolic deoxynucleotide concentrations are high due to the activity of the de novo synthesis pathway, sufficient phosphorylated deoxyguanosine and deoxyadenosine are able to enter the mitochondrial matrix to sustain mtDNA synthesis in deoxyguanosine kinase deficient cells. At other phases of the cell cycle, however, when cytosolic deoxyribonucleotide concentrations are lower due to the absence of the de novo synthesis, insufficient phosphorylated deoxyguanosine and deoxyadenosine enter the mitochondrial matrix to support normal mtDNA synthesis in deoxyguanosine kinase-deficient cells.

The Br-dU labelling results explain our Southern blot data. Whenever a deoxyguanosine kinase-deficient fibroblast passes through S-phase, mtDNA levels are restored and, therefore, levels are stable as long as the cells are grown exponentially (Fig. 1). When cell cycling is inhibited, however, mtDNA levels drop in deoxyguanosine kinase-deficient fibroblasts (Fig. 4). Interestingly, mtDNA does not fully deplete in quiescent, deoxyguanosine kinase-deficient fibroblasts, but mtDNA levels seem to stabilize at around 20% of the level found in control cells (Fig. 4B). A faint, punctate Br-dU incorporation pattern was seen in the cytoplasm of deoxyguanosine kinase-deficient fibroblasts that were not in S-phase (Fig. 2D), suggesting that low levels of mtDNA synthesis occur in these cells. These observations are consistent with the possibility that low levels of dGMP and dAMP synthesized by deoxycytidine kinase in the cytosol are utilized as precursors for mtDNA synthesis, but import levels are insufficient to sustain normal mtDNA levels.

Although deoxyguanosine kinase is thought to be constitutively expressed throughout the cell cycle and in all tissues (2), deficiency of the enzyme leads to developmental- and tissue-specific mtDNA depletion. The unremarkable fetal development of patients with deoxyguanosine kinase deficiency may in part be explained by the high rate of cell proliferation and the associated high levels of de novo synthesis of deoxynucleotides. After birth, when cell proliferation progressively declines, deoxycytidine kinase may partly compensate for the deoxyguanosine kinase deficiency. Deoxycytidine kinase activity is known to be very low in adult liver and brain (27,28). Therefore, deoxyguanosine kinase deficiency may selectively affect these tissues after birth. The results also suggest that deoxyguanosine kinase is a potential regulator of mtDNA copy number in some tissues. Substrate limitation has been put forward as a regulator of mtDNA copy number by others (29), based on their observations that cells maintain a certain mass of mitochondrial genomes, irrespective of the size of the mtDNA molecules and number of replication origins.

It has been proposed that mitochondrial gene dosage may play an important role in the regulation of expression levels of mtDNA-encoded proteins (30). While our Southern blot analysis of control fibroblasts indicated that mtDNA levels were stable 0–40 days after serum withdraw (Fig. 4), our western blot results showed that the levels of mtDNA-encoded subunit II of cytochrome-c oxidase decreased during this period (Fig. 5). Thus, it appears that there is no direct correlation between mtDNA content and cytochrome-c oxidase content during the transition from cycling to resting cells. Interestingly, levels of the nuclear-encoded flavoprotein of succinate dehydrogenase remained stable after serum withdraw, suggesting that the stoichiometry of the respiratory chain enzyme complexes changes during the transition. Further experiments are necessary to determine whether the decline in cytochrome-c oxidase subunit II levels are regulated at the transcriptional or post-transcriptional level and what the effect of cell cycle arrest is on enzyme activity.

The key finding of this paper is the Southern blot hybridization result of fibroblasts cultured in serum-deprived medium (Fig. 4). The blot shows that, although mtDNA depletes in quiescent, deoxyguanosine kinase deficient fibroblasts, depletion can be prevented by dGMP and dAMP supplementation. These data are corroborated by western blot experiments which showed a steeper drop of cytochrome-c oxidase subunit II levels in the patient culture than in the control and supplemented patient cultures during serum withdraw (Fig. 5). The Southern blot results prove conclusively that deoxyguanosine kinase deficiency causes mtDNA depletion by restricting the dGMP and dAMP pools. It remains to be established whether these exogenously added purine deoxyribonucleoside monophosphates are further phosphorylated in the cytosol prior to mitochondrial import, or are imported directly from the cytosol and converted in the mitochondrial matrix. Mitochondrial inner membrane carriers that transport dGMP or dAMP efficiently have not been identified.

The deoxynucleotide supplementation results raise the issue of possible therapeutic applications, especially because no treatment of hepatocerebral mtDNA depletion syndrome is currently available. One must take into account, however, that so far we have only demonstrated bypass of deoxyguanosine kinase deficiency in a cell culture model and that perturbations of deoxyribonucleotide pools are potentially mutagenic (1,31). Further research of a mouse knock-out model of deoxyguanosine kinase deficiency is required to evaluate the uptake, therapeutic value and possible side effects of deoxynucleotide administration. These studies are now in progress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell cultures and growth conditions
A skin biopsy was obtained from a patient who carried a homozygous nonsense mutation in exon 3 of DGUOK (16). The patient displayed a similar clinical course as described for his brother with the same homozygous DGUOK mutation and hepatocerebral mtDNA depletion syndrome (13). Control skin biopsies were taken from children younger than 3 years undergoing orthopaedic surgery. Informed parental consent, in accordance with the guidelines of the participating institutions, was obtained for all biopsies. Primary fibroblast cultures were established according to standard techniques. Cells were routinely cultured in Dulbecco's modified Eagle's medium that included 862 mg/l of Glutamax I, 110 mg/l of sodium pyruvate and 4.5 g/l of glucose (Gibco, Invitrogen Ltd, Paisley, UK), and was supplemented with 100 µl/ml of fetal bovine serum, 50 U/ml of penicillin, 50 µg/ml of streptomycin and 50 µg/ml of uridine. Cell cycling was inhibited by culturing in routine medium without fetal bovine serum. Br-dU labelling experiments revealed that 0.5–1% of the control fibroblasts passed through S-phase in serum-deprived medium during a 16 h pulse. In some experiments, the serum-deprived patient cell culture was supplemented with 200 µM dGMP and 200 µM dAMP.

Cells were routinely cultured in plastic dishes at 37°C in a humidified atmosphere of 8% CO2 in air, but cultured onto glass coverslips for 2–3 days for immunocytochemical investigations. Medium was changed every 4 days. Cultures were checked for mycoplasma infection prior to all experiments.

Cytochemical staining and imaging procedures
Immunocytochemistry with anti-cytochrome-c oxidase subunit I monoclonal antibodies (Molecular Probes Inc., Eugene, OR, USA) (32) and the mitochondrion-selective dye Mitotracker Red CM-H2XRos (Molecular Probes Inc.) was performed essentially as reported elsewhere (7), except that primary antibodies were detected with 20 µg/ml of Alexa 488 goat anti-mouse IgG conjugate (Molecular Probes Inc.) and nuclei were counterstained with 1 µg of DAPI per ml of Citifluor-glycerol-PBS solution (Agar Scientific Ltd, Stansted, UK) during mounting.

To detect newly synthesized DNA, cells were cultured in routine medium in the presence of 15 µM Br-dU for 6 h. Coverslips were subsequently washed in phosphate-buffered saline (PBS), incubated for 20 min at -20°C in 50 mM glycine–HCl (pH 2.0) containing 70% ethanol, and washed again in PBS. Following this, coverslips were incubated for 45 min at 37°C in a humidified atmosphere with anti-Br-dU antibodies and nucleases in a PBS-glycerol solution (from the 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit I; Roche Diagnostics Ltd, Lewes, UK), 10-fold diluted in 66 mM Tris-buffer (pH 8.0), containing 660 µM MgCl2 and 1 mM ß-mercaptoethanol. After primary antibody incubation, coverslips were washed with PBS, incubated for 45 min at 37°C in a humidified atmosphere with 20 µg/ml of Alexa 488 goat anti-mouse IgG conjugate in PBS containing 2% normal goat serum, and washed again. Coverslips were mounted and cells were counterstained with DAPI as indicated above.

Image analysis was carried out using a Zeiss Axiophot epifluorescence microscope fitted with a motorized stage, autofocus and filter wheel. Images of triple-labelled fluorescent samples were captured using a Hamamatsu ORCA cooled integrating camera and analysed using a Zeiss KS400 image analysis system. Three integrated images (red, green and blue) were captured separately as grey-scale images, and were enhanced and segmented. Cell boundaries were defined to produce a binary mask of individual cells. A mitochondrial mask was created to ensure that only those areas covered by mitochondria within cells was analysed. Densitometric analysis was carried out on individual cells; high-resolution mean grey level measurements were recorded for both the antibody and Mitotracker images. The ratio of the mean grey levels of the antibody and Mitotracker images was calculated for each cell. Digitally enhanced pictures of the merged, pseudo-coloured grey images were produced to indicate staining. Graphic image processing was carried out with Zeiss ZS400 and Adobe Photoshop software.

Analysis of cell homogenates
Cell cultures were harvested by trypsinization and washed twice with PBS. Spectrophotometric cytochrome-c oxidase and citrate synthase activity assays were carried out with freeze-thawed, homogenized fibroblasts as described (33). DNA extractions and Southern blot hybridizations were performed exactly as documented elsewhere (9). Nuclear and mtDNA signals were quantified by phosphorimaging (9) and the ratio of these values was calculated for each lane to correct for uneven sample loading. Western blot analysis with monoclonal antibodies directed against subunit II of cytochrome-c oxidase (Molecular Probes Inc.) (32) and the flavoprotein of succinate dehydrogenase (Molecular Probes Inc.) (34) was carried out as described in Taanman et al. (7), using 1.5% n-dodecyl-ß-D-maltoside (Anatrace Inc., Maumee, OH, USA) cell extracts, except that 12.5% polyacrylamide, 5.5 M urea mini-gels were used. Signals on the film were quantified using the NIH Scion Image application.


   ACKNOWLEDGEMENTS
 
We thank Will Roberts for expert technical assistance in the immunocytochemical experiments and Siôn L. Williams for critical reading of the manuscript.


   FOOTNOTES
 
* To whom correspondence should be addressed at: University Department of Clinical Neurosciences, RFUCM, UCL, Rowland Hill Street, London NW3 2PF, UK. Tel: +44 2077940500, ext 5354; Fax: +44 2074726829; Email: j.taanman{at}rfc.ucl.ac.uk Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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